title: Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs).
abstract: Although the identification of the multigene family encoding mammalian olfactory receptors were identified more than 20 years ago, we are far from understanding olfactory perception because of the difficulties in functional expression of these receptors in heterologous cell systems. Cell-free (CF) or in vitro expression systems offer an elegant alternative route to cell based protein expression, as the functional expression of membrane proteins can be directly achieved from the genetic template without the need of cell cultivation and protein isolation. Here we investigated in detail the cell-free expression and membrane insertion of the olfactory receptor OR5 in dependence of different experimental conditions like probing different origins of the cell-free expression system (from bacteria, via plants and insects toward mammalian system) and lipid composition of the respective extracts. We provided substantial biochemical indications by radioactive labeling based on [(35)S]-methionine, followed by proteolytic digestion, and we found that the insertion of the olfactory receptor OR5 into liposomes resulted in an unidirectional orientation with the binding side exposed into the aqueous space, resembling the native orientation in the cilia of the olfactory neurons. We report the different results in synthesis capacity for the different in vitro systems employed as we like to demonstrate the first in vitro kit toward and ex situ and ex vivo odorant receptor array.
authors.affiliation: Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz 55128, Germany. ritz@mpip-mainz.mpg.de
authors.forename: Sandra
authors.initials: S
authors.lastname: Ritz
authors.forename: Michael
authors.initials: M
authors.lastname: Hulko
authors.forename: Christian
authors.initials: C
authors.lastname: Zerfass
authors.forename: Sylvia
authors.initials: S
authors.lastname: May
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Hospach
authors.forename: Nadejda
authors.initials: N
authors.lastname: Krasteva
authors.forename: Gabriele
authors.initials: G
authors.lastname: Nelles
authors.forename: Eva K
authors.initials: EK
authors.lastname: Sinner
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D008715:Methionine / Q000378:metabolism; D018034:Olfactory Receptor Neurons / Q000737:chemistry* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011817:Rabbits; D051381:Rats; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012156:Reticulocytes / Q000737:chemistry / Q000378:metabolism; D013462:Sulfur Radioisotopes; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: C113013:OR5 protein, rat; D018035:Receptors, Odorant; D013462:Sulfur Radioisotopes; D053835:Unilamellar Liposomes; D008715:Methionine
keywords: Cell-free protein synthesis; Olfactory receptor OR5; Proteolytic digestion assay; Rabbit reticulocyte lysate; Unidirectional membrane insertion